Journal of Chromatography B, 958 (2014) 75–82
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Liquid chromatography–tandem mass spectrometry method for toxicokinetics, tissue distribution, and excretion studies of T-2 toxin and its major metabolites in pigs Yongxue Sun 1 , Guijun Zhang 1 , Haiyan Zhao, Jianlong Zheng, Fangyuan Hu, Binghu Fang ∗ Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
a r t i c l e
i n f o
Article history: Received 28 June 2013 Accepted 8 March 2014 Available online 19 March 2014 Keywords: T-2 toxin LC–MS/MS Toxicokinetics Tissue distribution Excretion Pigs
a b s t r a c t A rapid and sensitive high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for quantitatively analyzing T-2 toxin and its major metabolites (HT-2 toxin and T-2 triol) in swine biological samples. For all matrices, liquid–liquid extraction (ethyl acetate or acetonitrile) and Varian Bond-Elut MycoSep cartridges for solid phase extraction were used for sample preparation. The analytes were separated via a Zorbax XDB-C18 column and were detected using LC–MS/MS with an electrospray ionization interface in positive ion mode. The resulting calibration curves offered satisfactory linearity (r2 > 0.992) within the test range. The limits of quantification for T-2 toxin, HT-2 toxin, and T-2 triol were 1 ng/mL (g/kg), 2 ng/mL (g/kg), and 5 ng/mL (g/kg), respectively. The recovery rates in different matrices ranged from 74.3% to 102.4%, and the interday and intraday precisions were all less than 10.2% for the target analytes. The developed method was successfully applied to toxicokinetics, tissue distribution, and excretion studies of T-2 toxin and its major metabolites after intravenous (i.v.) administration in pigs. The results provide important information for evaluating and controlling human exposure to residual T-2 toxin and its major metabolites in animal-derived food. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The contamination of foods and feeds with mycotoxins is a significant problem worldwide. T-2 toxin, one of the most acutely toxic trichothecenes produced by various Fusarium species, is a widely distributed contaminant in different crops for human and animal consumption [1,2]. It causes emesis, diarrhea, lethargy, weight loss, hemorrhage, immunosuppression, necrosis, cartilage damage, apoptosis, and death [3–5]. T-2 toxin is considered as one of the most dangerous contaminants by the European Food Safety Authority. T-2 toxin is rapidly transformed into numerous metabolites mainly through hydrolysis, hydroxylation, de-epoxidation, and conjugation, which produces HT-2 toxin and T-2 triol as major metabolites in different animals [6–8]. The available studies on T-2 toxin in different species, such as dogs, broiler chickens, cows, rats, and pigs, focus on the parent toxin, but information on the major metabolites is limited [9–14]. The consumption of contaminated products may transfer T-2 toxin and its metabolites into edible
animal tissues. Thus, understanding the bioavailability and fate of T-2 toxin in food-producing animals is of great importance. Several methods have been developed for simultaneously determining T-2 toxin and its major metabolites HT-2 toxin and T-2 triol in various matrices using gas chromatography with flame ionization detection, which requires sample derivatization [15] and enzyme-linked immunosorbent assay with poor selectivity [16]. Therefore, to overcome these drawbacks, establishing a sensitive and rapid liquid chromatography tandem mass spectrometry (LC–MS/MS) method for determining target compounds in biological samples is critical. This analytical method was successfully applied for the first time in toxicokinetics, tissue distribution, and excretion studies on T-2 toxin and its major metabolites after intravenous (i.v.) administration in pigs. The results could provide important information for both evaluating and controlling human exposure to residual T-2 toxin and its major metabolites in animalderived food. 2. Materials and methods 2.1. Chemicals and reagents
∗ Corresponding author. Tel.: +86 2085280665; fax: +86 2085284896. E-mail address: [email protected] (B. Fang). 1 The authors contributed equally to this work. http://dx.doi.org/10.1016/j.jchromb.2014.03.010 1570-0232/© 2014 Elsevier B.V. All rights reserved.
The analytical standards for T-2 toxin (98.0%), HT-2 toxin (99.9%), and T-2 triol (99.9%) were obtained from Sigma–Aldrich
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(St. Louis, MO, USA). HPLC grade acetonitrile, methanol, and ammonium acetate were purchased from Fisher Scientific Co. (Pittsburgh, PA, USA). Ethyl acetate and other reagents were at least analytical grade and supplied by Guangzhou Chemical Reagent Factory (Guangzhou, China). Bond Elut Mycotoxin solid phase extraction (SPE) cartridge (500 mg, 3 mL) was purchased from Varian Inc. (Palo Alto, CA, USA). Ultrapure water was prepared using a Milli-Q water purification system (EMD Millipore, Billerica, MA). 2.2. Instrumentation An Agilent 1200 series high-performance liquid chromatograph (Agilent, CA, USA) coupled with an Applied Biosystems API 4000 triple quadrupole mass spectrometer with an electrospray ionization (ESI) interface (Foster City, CA, USA) was used. Chromatographic separation was achieved on a Zorbax XDB-C18 column (150 mm × 2.1 mm, 3.5 m; Agilent, USA). Data acquisition and processing were performed using the Analyst® 1.5 software from Applied Biosystems. Universal 32R Centrifuge was purchased from Hettich Inc. (Tuttlingen, Germany). 2.3. Animals Twenty-eight healthy cross-bred (6.8 kg ± 0.5 kg, Duroc × Landrace × Yorkshire) pigs weighing 6.3–7.3 kg were purchased from Lizhi Agricultural Co., Ltd. (Guangzhou, China). The pigs were placed under controlled temperature (25 ◦ C) and humidity (45%). During the whole experiment the pigs were supplied with T-2 toxin-free commercial feed ad libitum. It was considered as blank feed after the presence of mycotoxin by a validated LC–MS/MS method, the LODs of T-2 toxin and its main metabolites were 0.1 g/kg [17]. All experimental pigs were housed under the aforementioned conditions for 1 week to acclimatize. All protocols were approved by the Institutional Animal Care Use Committee of South China Agricultural University. 2.4. Standard and sample preparation 2.4.1. Preparation of stock and working solutions The stock solutions of T-2 toxin (1 mg/mL), HT-2 toxin (0.5 mg/mL), and T-2 triol (0.5 mg/mL) were prepared with acetonitrile. Stock standard solutions were stored for 2 months. Working mixed standard solutions were prepared weekly by diluting the stock solution with a mixture of water and acetonitrile (50:50, v/v). All solutions were kept in dark and stored at 4 ◦ C. 2.4.2. Preparation of quality control (QC) samples A series of matrix standard solutions (1, 2, 5, 10, 20, 50, 100, and 200 ng/mL for T-2 toxin; 1, 2, 5, 10, 20, 50, 100, and 200 ng/mL for HT-2 toxin; and 5, 10, 20, 50, 100, 200, and 500 ng/mL for T2 triol) was prepared by spiking 800 L of blank biological matrix (plasma, fat, muscle, stomach, brain, small intestines, heart, lung, spleen, urine and feces) with 200 L of mixed working solutions of different concentrations as mentioned in Section 2.4.1. The calibration working solutions and QC samples were freshly prepared before use. 2.4.3. Sample pretreatment Conventional liquid–liquid extraction and SPE methods were applied to extract T-2 toxin, HT-2 toxin, and T-2 triol from biological samples (plasma, tissue homogenates (fat, muscle, stomach, brain, small intestines, heart, lung, and spleen), and urine. These samples were obtained from storage (−20 ◦ C) and thawed at room temperature. Feces were vacuum freeze-dried before pretreatment. Plasma extraction and urine extraction. Aliquots of plasma or urine (0.5 mL) were mixed with 0.5 mL acetonitrile. The sample was
vortexed for 30 s and centrifuged at 16,000 × g for 10 min at 4 ◦ C, and the supernatant was filtered through a 0.22 m syringe filter for LC–MS/MS analysis. Tissue extraction and feces extraction. Tissue and feces samples (2 g) were mixed with 5 mL of ethyl acetate in a polypropylene tube. The mixture was homogenized for 2 min, shaken for 20 min, and centrifuged at 6900 × g for 10 min at 4 ◦ C. Then, the supernatant was transferred and saved. The extraction was repeated once more (but not homogenized). The supernatants were combined and then applied to a Varian Bond-Elut Mycotoxin SPE cartridge, which was previously conditioned with 3 mL of methanol and 3 mL of ethyl acetate. The filtrate was aspirated at no more than 1.5 mL/min, collected in a glass tube, and evaporated to dryness under nitrogen at 40 ◦ C. Then, the residue was reconstituted in 1 mL of 20% acetonitrile in water, vortexed for 1 min, and centrifuged for 16,000 × g for 10 min at 4 ◦ C. Finally, the supernatant was filtered through a 0.22 m syringe filter and injected for LC–MS/MS analysis. 2.5. Chromatographic conditions The mobile phase was delivered at a flow rate of 0.25 mL/min using a gradient elution profile consisting of 5.0 mM ammonium acetate in water (A) and acetonitrile (B). The gradient elution was performed as follows: 0–1.0 min 5–60%B, 1.0–6.5 min 60–5%B, 6.5–12.0 min 5%B. The injection volume was 5 L. 2.6. Mass spectrometer conditions Analytes were detected by MS/MS with an ESI interface in positive multiple reaction monitoring (MRM) mode. The ESI-MS/MS operating parameters used in this study were as follows: ion spray voltage, 5.0 kV; source temperature, 650 ◦ C; curtain gas, 20 psi; and ion source gas 1 and gas 2 at 65 psi and 60 psi, respectively. The mass transitions of T-2 toxin (m/z 484.3 > 305.2, 484.3 > 215.1), HT-2 toxin (m/z 442.3 > 215.2, 442.3 > 263.2), and T-2 triol (m/z 400.3 > 215.1, 400.3 > 125.2) were optimized. The declustering potentials were 54, 54, and 45, respectively. The collision energy was set to 19 and 24 for T-2 toxin, 21 and 20 for HT-2 toxin, and 17 and 19 eV for T-2 triol. 2.7. Method validation A thorough and complete method validation for assaying T-2 toxin, HT-2 toxin, and T-2 triol in biological matrices was performed according to the Commission Decision 2002/657/EC [17]. The validation parameters included selectivity, linearity, recovery and precision, limit of detection (LOD), limit of quantification (LOQ), matrix effect (ME), and stability. 2.7.1. Selectivity The selectivity of the method was determined by measuring the level of interfering substances in different sources of blank biological matrix. 2.7.2. Linearity, LOD, and LOQ The matrix-matched calibration curves were performed via linear regression of the peak area of T-2 toxin, HT-2 toxin, and T-2 triol (X-axis) and the nominal standard concentrations (1, 2, 5, 10, 20, 50, 100, and 200 ng/mL (g/kg) for T-2 toxin; 1, 2, 5, 10, 20, 50, 100, and 200 ng/mL (g/kg) for HT-2 toxin; and 5, 10, 20, 50, 100, 200, and 500 ng/mL (g/kg) for T-2 triol) (Y-axis) with a 1/X2 weighting factor, described as y = ax + b. The concentrations of the QCs and samples were calculated using the regression equation of the calibration curve. LOD was calculated as the analyte concentration that produced a peak signal three times the background noise from the
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chromatogram, whereas the LOQ was the analyte concentration that produced a peak signal 10 times the background noise. And that LOQ is the lowest point for which the accuracy and precision is investigated and proven. 2.7.3. Trueness and precision The trueness and precision of the method were evaluated from spiked biological matrices at three concentration levels (LOQ, 10, 100 ng/mL (g/kg) for T-2 toxin, LOQ, 20, 200 ng/mL (g/kg) for HT-2 toxin, and LOQ, 50, 500 ng/mL (g/kg) for T-2 triol) with six replicates at each level for three consecutive days. The determinations was carried out by comparing the response of the target analytes in biological matrices after extraction to the response of the same concentration of each analyte spiked into the solution extracted from blank biological matrices. 2.7.4. Matrix effect (ME) The absolute ME was evaluated by comparing the peak areas of the standard spiked extract with the corresponding peak area of the standard solution [19]. An ME value of 100% indicates that no ME was present. ME values 100% indicate signal enhancement. 2.7.5. Stability The analyte stability of the swine biological samples was investigated by determinations of QC samples at different concentrations. For short-term temperature stability, the QC samples were thawed at room temperature and stored at room temperature for 24 h prior to analysis. For freeze–thaw stability, the QC samples were stored at −20 ◦ C for 24 h and thawed at room temperature. Then, the samples were refrozen for 24 h under the same conditions. After three cycles, the concentrations of T-2 toxin, HT-2 toxin, and T-2 triol were evaluated, and the stability was calculated by comparing the concentrations with those obtained before freezing. 2.8. Experimental design We applied the bioanalytical method developed and validated previously to study toxicokinetics, tissue distribution, and excretion of T-2 toxin and its major metabolites in pigs. Twenty-eight pigs were randomly divided into four groups. Group A (six pigs) received T-2 toxin intravenously as a single dose at 1 mg/kg BW to study the toxicokinetics of T-2 and its major metabolites HT-2 and T-2 triol. Group B (12 pigs) was used to study the tissue distribution of T-2 toxin and its major metabolites, which were also given T-2 toxin intravenously as a single dose rate at 1 mg/kg BW. Pigs of group C (seven pigs) received i.v. administration T-2 toxin as a single dose at 0.5 mg/kg BW to study the excretion of T-2 and its major metabolites. The pigs in group D (three pigs) did not receive any treatment and they were used to determine the validation criteria for the analytical method. The solutions for i.v. administration were prepared daily by dissolving the T-2 toxin standard in ethyl alcohol - sterilized deionized water (1:1). For groups A, B, and C, the T-2 toxin was administered intravenously into the ear vein of pigs using a catheter. The pigs were fasted from12 h before drug administration until 6 h after administration. 2.8.1. Toxicokinetics Blood samples (2 mL) were collected in heparinized tube from the jugular vein of each pig in group A before and at 3, 6, 10, 15, 20, 30, and 45 min as well as 1, 1.5, 2, and 3 h after i.v. administration of the T-2 toxin. Blood samples were centrifuged (1500 × g for 10 min), and plasma was harvested and stored frozen at −20 ◦ C until analysis.
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2.8.2. Tissue distribution studies The pigs in group B were euthanized using carbon dioxide at 0.5, 1, 3, and 6 h (n = 3) after i.v. administration of T-2 toxin. The swine were immediately exsanguinated, and samples (2 g) from the heart, liver, spleen, lungs, kidneys, stomach, small intestines, muscles, brain, and fat tissues were separately collected. Each of the tissue specimens was carefully weighed and homogenized in saline solution. The obtained tissues were immediately stored at −20 ◦ C until LC–MS/MS analysis. 2.8.3. Excretion studies The pigs in group C were administered T-2 toxin intravenously as a single dose at 0.5 mg/kg BW. Then, the pigs were individually placed in stainless steel metabolic cages to allow the separate collection of urine and feces samples. These samples were collected at 0–4 h, 4–8 h, 8–12 h, 12–24 h, 24–36 h, 1.5–2 d, 2–3 d, 3–4 d, and 4–6 d post-dosing. The volume of urine samples and the weight of dry feces samples were recorded. The urine and feces samples were stored at −20 ◦ C until analysis by LC–MS/MS. 2.9. Calculation The concentration–time curves were analyzed using Microsoft Excel 2007 (Microsoft Co., USA). Data were expressed as mean ± S.D. The toxicokinetics study of T-2 toxin and its major metabolites was performed using a non-compartmental model of WinNonlin Program (version 5.2; Pharsight Corporation, Mountain View, CA, USA). The tissue distribution was calculated using linear regression analysis of the concentration–time curve. The elimination rate constant (z) was the slope of linear regression equation on log-transferred residue concentration (ln C) against time. The terminal elimination half-lives (t1/2z ) were calculated from the equation: t1/2z = 0.693 z−1 . The regulation of excretion was calculated as the excretion volume of T-2 toxin and its major metabolites at every time quantum (Q), Q = C × V, where C is the average concentration of the poison and V is the total volume of urine during each time quantum. 3. Results and discussion 3.1. Method development 3.1.1. Optimization of extraction conditions Methods for extracting T-2 toxin, HT-2 toxin, and T-2 triol from the biological matrix usually rely on toxin structure. Ethyl acetate, methanol, dichloromethane, and a mixture of acetonitrile/water are often used as extraction solvents for simultaneous extraction and determination of trichothecenes including T-2 toxin [20]. Several extraction solvents, such as ethyl acetate and acetonitrile, were tested. Acetonitrile was the best among the solvents because of the higher extraction recovery and less matrix interferences for T-2 toxin, HT-2 toxin, and T-2 triol in plasma and urine samples. However, the relative low recovery rates (90%). Cleanup is an essential step in complex matrices because the purity of the sample affects the sensitivity of the results, especially at trace levels. Varian Bond-Elut MycoSep cartridges, which contain charcoal, alumina, and celite, were frequently used for T-2 toxin purification and resulted in high recoveries and a lower limit of detection. 3.1.2. Optimization of LC conditions The composition of the mobile phase is critical in achieving good chromatographic behavior and appropriate ionization for
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Fig. 1. Representative MRM chromatograms of blank plasma (A), plasma spiked with T-2 toxin, HT-2 toxin and T-2 triol at level of 10 ng/mL (B), and real plasma sample (C) obtained 10 min after a single i.v. administration of T-2 toxin (1.0 mg/kg BW).
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optimal LC separation. Several kinds of mobile phases, including acetonitrile–ammonium acetate (5 mM), acetonitrile–water (0.1% formic acid), and methanol–water (0.1% formic acid), were investigated. The results showed that acetonitrile–ammonium acetate (5 mM) achieves satisfactory peaks and good sensitivity. Thus, this mobile phase was chosen with a Zorbax XDB-C18 column in the present study. 3.1.3. Optimization of MS conditions ESI was used to achieve good sensitivity and fragmentation. T-2 toxin, HT-2 toxin, and T-2 triol exhibited stronger signal responses and lower background noise in positive ion mode. The other parameters of the MS/MS for the analysis, such as spray voltage, declustering potential, and collision energy, were optimized through manual manipulation to provide the best response. In our method, one precursor ion and two product ions were monitored for the target analytes. The most intense and stable fragment ion was used for quantification, which allowed us to reach four IPs according to the Commission Decision 2002/657/EC [18]. 3.2. Method validation 3.2.1. Selectivity The representative ion chromatograms of blank plasma, spiked plasma with three toxins, and real plasma samples are shown in Fig. 1. The results demonstrate no interfering peaks within the 2.5% margin of the relative retention times of T-2 toxin (m/z 484.3 > 305.2, 7.8 min), HT-2 toxin (m/z 442.3 > 215.2, 6.5 min), and T-2 triol (m/z 400.3 > 215.1, 5.5 min) in the MRM chromatograms. Moreover, the results also indicate the absence of endogenous substances in other blank biological samples. 3.2.2. Linearity, LOD, and LOQ The matrix-matched calibration curves were obtained at concentrations ranging from 1 to 200 ng/mL (g/kg) for T-2 toxin, from 2 to 200 ng/mL (g/kg) for HT-2 toxin, and from 5 to 500 ng/mL (g/kg) for T-2 triol in all biological matrices. The results show good linearity (r2 > 0.992) over the concentration ranges tested in all biological matrices. The LOD was determined as the concentration at a signal-to-noise ratio of 3 and LOQ is the concentration at a signalto-noise ratio of 10. The LODs for T-2 toxin, HT-2 toxin, and T-2 triol in all biological matrices were 0.3, 0.6, and 2 ng/mL (g/kg), and the LOQs were 1, 2, and 5 ng/mL (g/kg), respectively. 3.2.3. Trueness and precision Trueness and precision for T-2 toxin, HT-2 toxin, and T-2 triol were determined at three levels (low, medium, and high) with six replicates at each level for three consecutive days in all biological matrices. As shown in Table 1, the recovery rates ranged from 74.3% to 102.4% for the target analytes, and the intraday and interday precision values (RSDs) were all less than 10.2%. The results, which were within the acceptable range for trueness and precision, proved that the analytical method was reliable and reproducible for the quantitative analysis of the three toxins in swine biological samples. 3.2.4. Matrix effect The ME determined by comparing the peak areas of the analyte standard solutions with those of the standard solutions spiked into blank biological matrices after extraction ranged from 79.3% to 93.8% in different biological matrices. The matrix-matched calibration curves were required for an accurate quantification of the analyte concentrations during ion suppression.
Fig. 2. Mean plasma concentration–time curves of T-2 toxin, HT-2 toxin, and T2 triol after a single i.v. administration of T-2 toxin (1 mg/kg BW) in pigs (n = 6). Results are presented as mean values + SD.
3.2.5. Stability The results of the stability test showed that T-2 toxin, HT-2 toxin, and T-2 triol were stable at room temperature for 24 h and through repeated freeze–thaw cycles. The results indicated that the established method for storage and intermittent analysis is suitable for large-scale sample analysis.
3.3. Plasma toxicokinetics The mean plasma concentration–time profiles of T-2 toxin, HT-2 toxin, and T-2 triol after a single i.v. administration (1 mg/kg BW) are presented in Fig. 2. The toxicokinetics parameters determined via the non-compartmental analysis of T-2 toxin, HT-2 toxin, and T-2 triol for i.v. administration are listed in Table 2. The plasma concentrations of T-2 toxin, HT-2 toxin, and T2 triol after i.v. administration were all detected until after 3 h, which indicated that the pigs absorbed and metabolized T-2 toxin. The mean Cmax were 2736 ± 236.3 ng/mL, 208.1 ± 25.3 ng/mL, and 116.3 ± 14.5 ng/mL, respectively. The T-2 toxin concentration was much higher than those of HT-2 toxin and T-2 triol, which caused T-2 toxicity and damaged the animal tissues [8]. Clinically, the body of pigs initially could appear persistent vomiting, trembling and odontoprisis at about 10 min after intravenous administration of T-2 toxin. A variety of clinical symptoms continued for about 40 min. Later, the pigs became clinically normal again within the experimental period. The areas under the concentration–time curves (AUC0–∞ ) of T2 toxin, HT-2 toxin, and T-2 triol were 42.53 ± 4.60 g min mL−1 , 13.13 ± 2.08 g min mL−1 , and 4.98 ± 0.25 g min mL−1 , respectively. The terminal elimination half-lives (t1/2z ) of these toxicants were 34.63 ± 2.95 min, 73.79 ± 5.53 min, and 159 ± 13.4 min, respectively. The t1/2z of T-2 toxin was longer than in dogs at 0.4 mg/kg BW (5.8 min), in broilers at 0.02 mg/kg BW (3.9 min), and in swine at 0.5 mg/kg BW (11.7 min) all after i.v. administrations [12–14]. The reason for this result is unknown but may be attributed to the differences in animal species, the LOQ of the analytical method, dosage of administration, body condition, age of the animals, and the system used for analysis. A low plasma clearance 0.025 ± 0.003 L/min/kg may also explain the relative tolerance of pigs to T-2 toxin. The t1/2z of HT-2 toxin (73.79 min) and T-2 triol (159 min) were longer than the parent toxin (34.63 min) because of the rapid deacetylation of T-2 toxin to metabolites. This result is in accordance with the previous study in dogs [12], which indicated that T-2 toxin, HT-2 toxin, and T-2 triol could provide important information for evaluating animal and human exposure to residual T-2 toxin.
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Table 1 Extraction recovery rates and intraday and interday precisions of the assay for T-2 toxin, HT-2 toxin, and T-2 triol in plasma, urine, liver, kidney, and fat. Sample matrix
Analyte
Spiked concentration ng/mL (g/kg)
Extraction recovery (%)
Plasma
T-2
1 10 100 2 20 200 5 50 500
92.3 95.2 101 95.4 92.2 89.3 90.1 93.2 96.3
± ± ± ± ± ± ± ± ±
4.09 3.78 4.53 3.79 3.16 3.71 4.31 3.28 3.02
4.4 4.0 4.5 3.9 3.4 4.2 4.8 3.5 3.1
9.3 7.9 8.2 9.3 8.2 9.6 9.2 8.5 7.3
1 10 100 2 20 200 5 50 500
93.1 91.2 89.3 92.3 91.7 88.3 92.1 94.2 100
± ± ± ± ± ± ± ± ±
3.45 3.49 2.91 2.98 3.36 2.81 3.46 3.29 2.40
3.7 3.8 3.3 3.2 3.7 3.2 3.8 3.5 2.4
8.4 7.3 6.9 10.2 9.4 10.0 9.8 8.1 7.3
1 10 100 2 20 200 5 50 500
85.1 80.2 83.7 75.9 81.3 83.2 79.4 81.2 83.6
± ± ± ± ± ± ± ± ±
3.62 2.85 3.33 2.12 2.39 3.12 4.21 3.15 3.63
4.3 3.6 4.0 2.8 2.9 3.8 5.3 3.9 4.3
7.9 6.8 8.3 9.5 8.3 7.9 8.1 7.7 9.3
1 10 100 2 20 200 5 50 500
83.1 81.0 86.3 77.4 80.6 85.6 82.1 80.5 87.6
± ± ± ± ± ± ± ± ±
2.92 2.52 3.27 3.87 3.05 3.27 2.35 2.56 4.53
3.5 3.1 3.8 5.0 3.9 3.8 2.9 3.2 5.2
8.3 7.9 9.2 9.4 7.0 8.2 6.4 7.4 10.1
1 10 100 2 20 200 5 50 500
75.3 78.3 75.3 75.4 78.8 76.6 74.3 77.3 74.2
± ± ± ± ± ± ± ± ±
3.03 2.64 3.42 2.72 4.21 3.12 2.52 2.03 2.45
4.0 3.4 4.5 3.6 5.3 4.1 3.4 2.6 3.3
8.3 9.7 8.1 7.3 9.9 8.4 8.2 9.1 8.6
HT-2
T-2 triol
Urine
T-2
HT-2
T-2 triol
Liver
T-2
HT-2
T-2 triol
Kidney
T-2
HT-2
T-2 triol
Fat
T-2
HT-2
T-2 triol
3.4. Tissue distribution studies Tissue distributions of T-2 toxin, HT-2 toxin, and T-2 triol were investigated in pigs following a single i.v. dose of T-2 toxin (1 mg/kg BW). The results (Fig. 3) indicated that T-2 toxin is rapidly metabolized into more polar metabolites of HT-2 toxin and T-2 triol. They underwent a rapid and wide distribution into tissues during the examination period. This result is in accordance with the
Intra-day (RSD %)
Inter-day (RSD %)
metabolism of T-2 toxin in tissues and gastrointestinal tract of swine [11]. T-2 toxin was not detected in the liver and kidney, most probably T-2 toxin was indeed distributed into these main eliminating organs, and it was extensively metabolized or excreted. This result is consistent with the findings that T-2 toxin was not detected in the liver after arterial injection of T-2 toxin at 1.2 mg/kg BW in pigs [10] and after oral administration of 3H T-2 toxin in mice [9].
Table 2 Main toxicokinetics parameters of T-2 toxin and its metabolites (HT-2 toxin and T-2 triol) after a single i.v. administration of T-2 toxin (1 mg/kg BW) in pigs. Results are presented as means ± SD. Toxicokinetic parameters
T-2 toxin
z (1/min) t1/2z (min) AUC0–∞ (g min mL−1 ) MRT (min) Cmax (ng/mL) Vss (L/kg) CL (L/min/kg)
0.021 34.63 42.53 22.06 2736 1.18 0.025
± ± ± ± ± ± ±
0.002 2.95 4.60 1.71 236.30 0.08 0.003
HT-2 toxin
T-2 triol
0.010 ± 73.79 ± 13.13 ± 94.56 ± 208.1 ± – –
0.005 ± 159 ± 4.98 ± 211.0 ± 116.3 ± – –
0.001 5.53 2.08 5.40 25.30
0.001 13.40 0.25 19.40 14.50
Notes. AUC0–∞ : the area under the plasma concentration–time curve; t1/2z : half-life of elimination, Cmax : maximum plasma concentration; Tmax : time to maximum plasma concentration, CL: clearance; Vss : volume of distribution; MRT: mean residence time; SD: standard deviation.
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Fig. 3. Tissue distribution of T-2 toxin, HT-2 toxin, and T-2 triol after a single i.v. administration of T-2 toxin (1.0 mg/kg BW) in pigs (n = 3). Results are presented as mean values + SD.
The highest T-2 toxin concentration was detected in fat tissues (58.6 ± 8.21 g/kg), followed by the lungs (54.0 ± 4.27 g/kg) and spleen (47.8 ± 5.70 g/kg). However, Beasley et al. [10] reported that fat could not be detected in pigs, which may be attributed to factors including the age of the animals, administration dosage, and body conditions. The levels of T-2 toxin in all tissues could not be detected at 6 h after dosing. The highest HT-2 toxin concentrations were also observed in the liver (216.3 ± 16.9 g/kg), followed by the kidney (206.3 ± 20.3 g/kg) and the small intestines (140.5 ± 9.2 g/kg). HT-2 toxin was still detected at 6 h after administration. T-2 toxin, HT-2 toxin, and T-2 triol were found at low concentrations in the brain, which suggest that the toxicants did not efficiently cross the blood–brain barrier. The results of T-2 triol were similar to that of HT-2 toxin in tissues, except its concentrations were much lower than that of HT-2 toxin. In summary, T-2 toxin is metabolized into an extensive range of metabolites such as HT-2 toxin, especially in the liver, kidney, and small intestines.
Given the lower toxicity and complex elimination characteristics of the metabolites than T-2 toxin, we just obtained the elimination half-lives (t1/2z ) of T-2 toxin. The mean log-transferred concentrations (ln C) of T-2 toxin in pig tissues over time were analyzed by linear regression analysis, except for the liver and kidney. The t1/2z of T-2 toxin in tissues including fat, muscle, stomach, brain, small intestines, heart, lung and spleen were 41.24 min, 33.12 min, 25.53 min, 22.64 min, 21.20 min, 16.32 min, 15.33 min, 11.71 min, respectively. According to the results, T-2 toxin has a longer t1/2z (41.24 min) in fat tissues, indicating its selective retention. The depletion of T-2 toxin in the pig spleen was much faster than other tissues. 3.5. Excretion studies The cumulative excretion of T-2 toxin, HT-2 toxin, and T-2 triol in urine and feces after a single i.v. administration of T-2 toxin (0.5 mg/kg BW) was determined. 4 h after administration of T-2
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Fig. 4. The quality of T-2 toxin, HT-2 toxin, and T-2 triol in urine in different time after a single i.v. administration of T-2 toxin (0.5 mg/kg BW) in pigs (n = 7). Results are presented as mean values + SD.
toxin, the T-2 toxin, HT-2 toxin, and T-2 triol concentrations in urines were 30.9 ± 2.1 ng/mL, 614.4 ± 177 ng/mL, 306 ± 70 ng/mL, respectively. The excretion data of T-2 toxin, HT-2 toxin, and T2 triol in urine indicated that merely
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Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb
Liquid chromatography–tandem mass spectrometry method for toxicokinetics, tissue distribution, and excretion studies of T-2 toxin and its major metabolites in pigs Yongxue Sun 1 , Guijun Zhang 1 , Haiyan Zhao, Jianlong Zheng, Fangyuan Hu, Binghu Fang ∗ Department of Veterinary Pharmacology and Toxicology, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642, China
a r t i c l e
i n f o
Article history: Received 28 June 2013 Accepted 8 March 2014 Available online 19 March 2014 Keywords: T-2 toxin LC–MS/MS Toxicokinetics Tissue distribution Excretion Pigs
a b s t r a c t A rapid and sensitive high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for quantitatively analyzing T-2 toxin and its major metabolites (HT-2 toxin and T-2 triol) in swine biological samples. For all matrices, liquid–liquid extraction (ethyl acetate or acetonitrile) and Varian Bond-Elut MycoSep cartridges for solid phase extraction were used for sample preparation. The analytes were separated via a Zorbax XDB-C18 column and were detected using LC–MS/MS with an electrospray ionization interface in positive ion mode. The resulting calibration curves offered satisfactory linearity (r2 > 0.992) within the test range. The limits of quantification for T-2 toxin, HT-2 toxin, and T-2 triol were 1 ng/mL (g/kg), 2 ng/mL (g/kg), and 5 ng/mL (g/kg), respectively. The recovery rates in different matrices ranged from 74.3% to 102.4%, and the interday and intraday precisions were all less than 10.2% for the target analytes. The developed method was successfully applied to toxicokinetics, tissue distribution, and excretion studies of T-2 toxin and its major metabolites after intravenous (i.v.) administration in pigs. The results provide important information for evaluating and controlling human exposure to residual T-2 toxin and its major metabolites in animal-derived food. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The contamination of foods and feeds with mycotoxins is a significant problem worldwide. T-2 toxin, one of the most acutely toxic trichothecenes produced by various Fusarium species, is a widely distributed contaminant in different crops for human and animal consumption [1,2]. It causes emesis, diarrhea, lethargy, weight loss, hemorrhage, immunosuppression, necrosis, cartilage damage, apoptosis, and death [3–5]. T-2 toxin is considered as one of the most dangerous contaminants by the European Food Safety Authority. T-2 toxin is rapidly transformed into numerous metabolites mainly through hydrolysis, hydroxylation, de-epoxidation, and conjugation, which produces HT-2 toxin and T-2 triol as major metabolites in different animals [6–8]. The available studies on T-2 toxin in different species, such as dogs, broiler chickens, cows, rats, and pigs, focus on the parent toxin, but information on the major metabolites is limited [9–14]. The consumption of contaminated products may transfer T-2 toxin and its metabolites into edible
animal tissues. Thus, understanding the bioavailability and fate of T-2 toxin in food-producing animals is of great importance. Several methods have been developed for simultaneously determining T-2 toxin and its major metabolites HT-2 toxin and T-2 triol in various matrices using gas chromatography with flame ionization detection, which requires sample derivatization [15] and enzyme-linked immunosorbent assay with poor selectivity [16]. Therefore, to overcome these drawbacks, establishing a sensitive and rapid liquid chromatography tandem mass spectrometry (LC–MS/MS) method for determining target compounds in biological samples is critical. This analytical method was successfully applied for the first time in toxicokinetics, tissue distribution, and excretion studies on T-2 toxin and its major metabolites after intravenous (i.v.) administration in pigs. The results could provide important information for both evaluating and controlling human exposure to residual T-2 toxin and its major metabolites in animalderived food. 2. Materials and methods 2.1. Chemicals and reagents
∗ Corresponding author. Tel.: +86 2085280665; fax: +86 2085284896. E-mail address: [email protected] (B. Fang). 1 The authors contributed equally to this work. http://dx.doi.org/10.1016/j.jchromb.2014.03.010 1570-0232/© 2014 Elsevier B.V. All rights reserved.
The analytical standards for T-2 toxin (98.0%), HT-2 toxin (99.9%), and T-2 triol (99.9%) were obtained from Sigma–Aldrich
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(St. Louis, MO, USA). HPLC grade acetonitrile, methanol, and ammonium acetate were purchased from Fisher Scientific Co. (Pittsburgh, PA, USA). Ethyl acetate and other reagents were at least analytical grade and supplied by Guangzhou Chemical Reagent Factory (Guangzhou, China). Bond Elut Mycotoxin solid phase extraction (SPE) cartridge (500 mg, 3 mL) was purchased from Varian Inc. (Palo Alto, CA, USA). Ultrapure water was prepared using a Milli-Q water purification system (EMD Millipore, Billerica, MA). 2.2. Instrumentation An Agilent 1200 series high-performance liquid chromatograph (Agilent, CA, USA) coupled with an Applied Biosystems API 4000 triple quadrupole mass spectrometer with an electrospray ionization (ESI) interface (Foster City, CA, USA) was used. Chromatographic separation was achieved on a Zorbax XDB-C18 column (150 mm × 2.1 mm, 3.5 m; Agilent, USA). Data acquisition and processing were performed using the Analyst® 1.5 software from Applied Biosystems. Universal 32R Centrifuge was purchased from Hettich Inc. (Tuttlingen, Germany). 2.3. Animals Twenty-eight healthy cross-bred (6.8 kg ± 0.5 kg, Duroc × Landrace × Yorkshire) pigs weighing 6.3–7.3 kg were purchased from Lizhi Agricultural Co., Ltd. (Guangzhou, China). The pigs were placed under controlled temperature (25 ◦ C) and humidity (45%). During the whole experiment the pigs were supplied with T-2 toxin-free commercial feed ad libitum. It was considered as blank feed after the presence of mycotoxin by a validated LC–MS/MS method, the LODs of T-2 toxin and its main metabolites were 0.1 g/kg [17]. All experimental pigs were housed under the aforementioned conditions for 1 week to acclimatize. All protocols were approved by the Institutional Animal Care Use Committee of South China Agricultural University. 2.4. Standard and sample preparation 2.4.1. Preparation of stock and working solutions The stock solutions of T-2 toxin (1 mg/mL), HT-2 toxin (0.5 mg/mL), and T-2 triol (0.5 mg/mL) were prepared with acetonitrile. Stock standard solutions were stored for 2 months. Working mixed standard solutions were prepared weekly by diluting the stock solution with a mixture of water and acetonitrile (50:50, v/v). All solutions were kept in dark and stored at 4 ◦ C. 2.4.2. Preparation of quality control (QC) samples A series of matrix standard solutions (1, 2, 5, 10, 20, 50, 100, and 200 ng/mL for T-2 toxin; 1, 2, 5, 10, 20, 50, 100, and 200 ng/mL for HT-2 toxin; and 5, 10, 20, 50, 100, 200, and 500 ng/mL for T2 triol) was prepared by spiking 800 L of blank biological matrix (plasma, fat, muscle, stomach, brain, small intestines, heart, lung, spleen, urine and feces) with 200 L of mixed working solutions of different concentrations as mentioned in Section 2.4.1. The calibration working solutions and QC samples were freshly prepared before use. 2.4.3. Sample pretreatment Conventional liquid–liquid extraction and SPE methods were applied to extract T-2 toxin, HT-2 toxin, and T-2 triol from biological samples (plasma, tissue homogenates (fat, muscle, stomach, brain, small intestines, heart, lung, and spleen), and urine. These samples were obtained from storage (−20 ◦ C) and thawed at room temperature. Feces were vacuum freeze-dried before pretreatment. Plasma extraction and urine extraction. Aliquots of plasma or urine (0.5 mL) were mixed with 0.5 mL acetonitrile. The sample was
vortexed for 30 s and centrifuged at 16,000 × g for 10 min at 4 ◦ C, and the supernatant was filtered through a 0.22 m syringe filter for LC–MS/MS analysis. Tissue extraction and feces extraction. Tissue and feces samples (2 g) were mixed with 5 mL of ethyl acetate in a polypropylene tube. The mixture was homogenized for 2 min, shaken for 20 min, and centrifuged at 6900 × g for 10 min at 4 ◦ C. Then, the supernatant was transferred and saved. The extraction was repeated once more (but not homogenized). The supernatants were combined and then applied to a Varian Bond-Elut Mycotoxin SPE cartridge, which was previously conditioned with 3 mL of methanol and 3 mL of ethyl acetate. The filtrate was aspirated at no more than 1.5 mL/min, collected in a glass tube, and evaporated to dryness under nitrogen at 40 ◦ C. Then, the residue was reconstituted in 1 mL of 20% acetonitrile in water, vortexed for 1 min, and centrifuged for 16,000 × g for 10 min at 4 ◦ C. Finally, the supernatant was filtered through a 0.22 m syringe filter and injected for LC–MS/MS analysis. 2.5. Chromatographic conditions The mobile phase was delivered at a flow rate of 0.25 mL/min using a gradient elution profile consisting of 5.0 mM ammonium acetate in water (A) and acetonitrile (B). The gradient elution was performed as follows: 0–1.0 min 5–60%B, 1.0–6.5 min 60–5%B, 6.5–12.0 min 5%B. The injection volume was 5 L. 2.6. Mass spectrometer conditions Analytes were detected by MS/MS with an ESI interface in positive multiple reaction monitoring (MRM) mode. The ESI-MS/MS operating parameters used in this study were as follows: ion spray voltage, 5.0 kV; source temperature, 650 ◦ C; curtain gas, 20 psi; and ion source gas 1 and gas 2 at 65 psi and 60 psi, respectively. The mass transitions of T-2 toxin (m/z 484.3 > 305.2, 484.3 > 215.1), HT-2 toxin (m/z 442.3 > 215.2, 442.3 > 263.2), and T-2 triol (m/z 400.3 > 215.1, 400.3 > 125.2) were optimized. The declustering potentials were 54, 54, and 45, respectively. The collision energy was set to 19 and 24 for T-2 toxin, 21 and 20 for HT-2 toxin, and 17 and 19 eV for T-2 triol. 2.7. Method validation A thorough and complete method validation for assaying T-2 toxin, HT-2 toxin, and T-2 triol in biological matrices was performed according to the Commission Decision 2002/657/EC [17]. The validation parameters included selectivity, linearity, recovery and precision, limit of detection (LOD), limit of quantification (LOQ), matrix effect (ME), and stability. 2.7.1. Selectivity The selectivity of the method was determined by measuring the level of interfering substances in different sources of blank biological matrix. 2.7.2. Linearity, LOD, and LOQ The matrix-matched calibration curves were performed via linear regression of the peak area of T-2 toxin, HT-2 toxin, and T-2 triol (X-axis) and the nominal standard concentrations (1, 2, 5, 10, 20, 50, 100, and 200 ng/mL (g/kg) for T-2 toxin; 1, 2, 5, 10, 20, 50, 100, and 200 ng/mL (g/kg) for HT-2 toxin; and 5, 10, 20, 50, 100, 200, and 500 ng/mL (g/kg) for T-2 triol) (Y-axis) with a 1/X2 weighting factor, described as y = ax + b. The concentrations of the QCs and samples were calculated using the regression equation of the calibration curve. LOD was calculated as the analyte concentration that produced a peak signal three times the background noise from the
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chromatogram, whereas the LOQ was the analyte concentration that produced a peak signal 10 times the background noise. And that LOQ is the lowest point for which the accuracy and precision is investigated and proven. 2.7.3. Trueness and precision The trueness and precision of the method were evaluated from spiked biological matrices at three concentration levels (LOQ, 10, 100 ng/mL (g/kg) for T-2 toxin, LOQ, 20, 200 ng/mL (g/kg) for HT-2 toxin, and LOQ, 50, 500 ng/mL (g/kg) for T-2 triol) with six replicates at each level for three consecutive days. The determinations was carried out by comparing the response of the target analytes in biological matrices after extraction to the response of the same concentration of each analyte spiked into the solution extracted from blank biological matrices. 2.7.4. Matrix effect (ME) The absolute ME was evaluated by comparing the peak areas of the standard spiked extract with the corresponding peak area of the standard solution [19]. An ME value of 100% indicates that no ME was present. ME values 100% indicate signal enhancement. 2.7.5. Stability The analyte stability of the swine biological samples was investigated by determinations of QC samples at different concentrations. For short-term temperature stability, the QC samples were thawed at room temperature and stored at room temperature for 24 h prior to analysis. For freeze–thaw stability, the QC samples were stored at −20 ◦ C for 24 h and thawed at room temperature. Then, the samples were refrozen for 24 h under the same conditions. After three cycles, the concentrations of T-2 toxin, HT-2 toxin, and T-2 triol were evaluated, and the stability was calculated by comparing the concentrations with those obtained before freezing. 2.8. Experimental design We applied the bioanalytical method developed and validated previously to study toxicokinetics, tissue distribution, and excretion of T-2 toxin and its major metabolites in pigs. Twenty-eight pigs were randomly divided into four groups. Group A (six pigs) received T-2 toxin intravenously as a single dose at 1 mg/kg BW to study the toxicokinetics of T-2 and its major metabolites HT-2 and T-2 triol. Group B (12 pigs) was used to study the tissue distribution of T-2 toxin and its major metabolites, which were also given T-2 toxin intravenously as a single dose rate at 1 mg/kg BW. Pigs of group C (seven pigs) received i.v. administration T-2 toxin as a single dose at 0.5 mg/kg BW to study the excretion of T-2 and its major metabolites. The pigs in group D (three pigs) did not receive any treatment and they were used to determine the validation criteria for the analytical method. The solutions for i.v. administration were prepared daily by dissolving the T-2 toxin standard in ethyl alcohol - sterilized deionized water (1:1). For groups A, B, and C, the T-2 toxin was administered intravenously into the ear vein of pigs using a catheter. The pigs were fasted from12 h before drug administration until 6 h after administration. 2.8.1. Toxicokinetics Blood samples (2 mL) were collected in heparinized tube from the jugular vein of each pig in group A before and at 3, 6, 10, 15, 20, 30, and 45 min as well as 1, 1.5, 2, and 3 h after i.v. administration of the T-2 toxin. Blood samples were centrifuged (1500 × g for 10 min), and plasma was harvested and stored frozen at −20 ◦ C until analysis.
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2.8.2. Tissue distribution studies The pigs in group B were euthanized using carbon dioxide at 0.5, 1, 3, and 6 h (n = 3) after i.v. administration of T-2 toxin. The swine were immediately exsanguinated, and samples (2 g) from the heart, liver, spleen, lungs, kidneys, stomach, small intestines, muscles, brain, and fat tissues were separately collected. Each of the tissue specimens was carefully weighed and homogenized in saline solution. The obtained tissues were immediately stored at −20 ◦ C until LC–MS/MS analysis. 2.8.3. Excretion studies The pigs in group C were administered T-2 toxin intravenously as a single dose at 0.5 mg/kg BW. Then, the pigs were individually placed in stainless steel metabolic cages to allow the separate collection of urine and feces samples. These samples were collected at 0–4 h, 4–8 h, 8–12 h, 12–24 h, 24–36 h, 1.5–2 d, 2–3 d, 3–4 d, and 4–6 d post-dosing. The volume of urine samples and the weight of dry feces samples were recorded. The urine and feces samples were stored at −20 ◦ C until analysis by LC–MS/MS. 2.9. Calculation The concentration–time curves were analyzed using Microsoft Excel 2007 (Microsoft Co., USA). Data were expressed as mean ± S.D. The toxicokinetics study of T-2 toxin and its major metabolites was performed using a non-compartmental model of WinNonlin Program (version 5.2; Pharsight Corporation, Mountain View, CA, USA). The tissue distribution was calculated using linear regression analysis of the concentration–time curve. The elimination rate constant (z) was the slope of linear regression equation on log-transferred residue concentration (ln C) against time. The terminal elimination half-lives (t1/2z ) were calculated from the equation: t1/2z = 0.693 z−1 . The regulation of excretion was calculated as the excretion volume of T-2 toxin and its major metabolites at every time quantum (Q), Q = C × V, where C is the average concentration of the poison and V is the total volume of urine during each time quantum. 3. Results and discussion 3.1. Method development 3.1.1. Optimization of extraction conditions Methods for extracting T-2 toxin, HT-2 toxin, and T-2 triol from the biological matrix usually rely on toxin structure. Ethyl acetate, methanol, dichloromethane, and a mixture of acetonitrile/water are often used as extraction solvents for simultaneous extraction and determination of trichothecenes including T-2 toxin [20]. Several extraction solvents, such as ethyl acetate and acetonitrile, were tested. Acetonitrile was the best among the solvents because of the higher extraction recovery and less matrix interferences for T-2 toxin, HT-2 toxin, and T-2 triol in plasma and urine samples. However, the relative low recovery rates (90%). Cleanup is an essential step in complex matrices because the purity of the sample affects the sensitivity of the results, especially at trace levels. Varian Bond-Elut MycoSep cartridges, which contain charcoal, alumina, and celite, were frequently used for T-2 toxin purification and resulted in high recoveries and a lower limit of detection. 3.1.2. Optimization of LC conditions The composition of the mobile phase is critical in achieving good chromatographic behavior and appropriate ionization for
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Fig. 1. Representative MRM chromatograms of blank plasma (A), plasma spiked with T-2 toxin, HT-2 toxin and T-2 triol at level of 10 ng/mL (B), and real plasma sample (C) obtained 10 min after a single i.v. administration of T-2 toxin (1.0 mg/kg BW).
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optimal LC separation. Several kinds of mobile phases, including acetonitrile–ammonium acetate (5 mM), acetonitrile–water (0.1% formic acid), and methanol–water (0.1% formic acid), were investigated. The results showed that acetonitrile–ammonium acetate (5 mM) achieves satisfactory peaks and good sensitivity. Thus, this mobile phase was chosen with a Zorbax XDB-C18 column in the present study. 3.1.3. Optimization of MS conditions ESI was used to achieve good sensitivity and fragmentation. T-2 toxin, HT-2 toxin, and T-2 triol exhibited stronger signal responses and lower background noise in positive ion mode. The other parameters of the MS/MS for the analysis, such as spray voltage, declustering potential, and collision energy, were optimized through manual manipulation to provide the best response. In our method, one precursor ion and two product ions were monitored for the target analytes. The most intense and stable fragment ion was used for quantification, which allowed us to reach four IPs according to the Commission Decision 2002/657/EC [18]. 3.2. Method validation 3.2.1. Selectivity The representative ion chromatograms of blank plasma, spiked plasma with three toxins, and real plasma samples are shown in Fig. 1. The results demonstrate no interfering peaks within the 2.5% margin of the relative retention times of T-2 toxin (m/z 484.3 > 305.2, 7.8 min), HT-2 toxin (m/z 442.3 > 215.2, 6.5 min), and T-2 triol (m/z 400.3 > 215.1, 5.5 min) in the MRM chromatograms. Moreover, the results also indicate the absence of endogenous substances in other blank biological samples. 3.2.2. Linearity, LOD, and LOQ The matrix-matched calibration curves were obtained at concentrations ranging from 1 to 200 ng/mL (g/kg) for T-2 toxin, from 2 to 200 ng/mL (g/kg) for HT-2 toxin, and from 5 to 500 ng/mL (g/kg) for T-2 triol in all biological matrices. The results show good linearity (r2 > 0.992) over the concentration ranges tested in all biological matrices. The LOD was determined as the concentration at a signal-to-noise ratio of 3 and LOQ is the concentration at a signalto-noise ratio of 10. The LODs for T-2 toxin, HT-2 toxin, and T-2 triol in all biological matrices were 0.3, 0.6, and 2 ng/mL (g/kg), and the LOQs were 1, 2, and 5 ng/mL (g/kg), respectively. 3.2.3. Trueness and precision Trueness and precision for T-2 toxin, HT-2 toxin, and T-2 triol were determined at three levels (low, medium, and high) with six replicates at each level for three consecutive days in all biological matrices. As shown in Table 1, the recovery rates ranged from 74.3% to 102.4% for the target analytes, and the intraday and interday precision values (RSDs) were all less than 10.2%. The results, which were within the acceptable range for trueness and precision, proved that the analytical method was reliable and reproducible for the quantitative analysis of the three toxins in swine biological samples. 3.2.4. Matrix effect The ME determined by comparing the peak areas of the analyte standard solutions with those of the standard solutions spiked into blank biological matrices after extraction ranged from 79.3% to 93.8% in different biological matrices. The matrix-matched calibration curves were required for an accurate quantification of the analyte concentrations during ion suppression.
Fig. 2. Mean plasma concentration–time curves of T-2 toxin, HT-2 toxin, and T2 triol after a single i.v. administration of T-2 toxin (1 mg/kg BW) in pigs (n = 6). Results are presented as mean values + SD.
3.2.5. Stability The results of the stability test showed that T-2 toxin, HT-2 toxin, and T-2 triol were stable at room temperature for 24 h and through repeated freeze–thaw cycles. The results indicated that the established method for storage and intermittent analysis is suitable for large-scale sample analysis.
3.3. Plasma toxicokinetics The mean plasma concentration–time profiles of T-2 toxin, HT-2 toxin, and T-2 triol after a single i.v. administration (1 mg/kg BW) are presented in Fig. 2. The toxicokinetics parameters determined via the non-compartmental analysis of T-2 toxin, HT-2 toxin, and T-2 triol for i.v. administration are listed in Table 2. The plasma concentrations of T-2 toxin, HT-2 toxin, and T2 triol after i.v. administration were all detected until after 3 h, which indicated that the pigs absorbed and metabolized T-2 toxin. The mean Cmax were 2736 ± 236.3 ng/mL, 208.1 ± 25.3 ng/mL, and 116.3 ± 14.5 ng/mL, respectively. The T-2 toxin concentration was much higher than those of HT-2 toxin and T-2 triol, which caused T-2 toxicity and damaged the animal tissues [8]. Clinically, the body of pigs initially could appear persistent vomiting, trembling and odontoprisis at about 10 min after intravenous administration of T-2 toxin. A variety of clinical symptoms continued for about 40 min. Later, the pigs became clinically normal again within the experimental period. The areas under the concentration–time curves (AUC0–∞ ) of T2 toxin, HT-2 toxin, and T-2 triol were 42.53 ± 4.60 g min mL−1 , 13.13 ± 2.08 g min mL−1 , and 4.98 ± 0.25 g min mL−1 , respectively. The terminal elimination half-lives (t1/2z ) of these toxicants were 34.63 ± 2.95 min, 73.79 ± 5.53 min, and 159 ± 13.4 min, respectively. The t1/2z of T-2 toxin was longer than in dogs at 0.4 mg/kg BW (5.8 min), in broilers at 0.02 mg/kg BW (3.9 min), and in swine at 0.5 mg/kg BW (11.7 min) all after i.v. administrations [12–14]. The reason for this result is unknown but may be attributed to the differences in animal species, the LOQ of the analytical method, dosage of administration, body condition, age of the animals, and the system used for analysis. A low plasma clearance 0.025 ± 0.003 L/min/kg may also explain the relative tolerance of pigs to T-2 toxin. The t1/2z of HT-2 toxin (73.79 min) and T-2 triol (159 min) were longer than the parent toxin (34.63 min) because of the rapid deacetylation of T-2 toxin to metabolites. This result is in accordance with the previous study in dogs [12], which indicated that T-2 toxin, HT-2 toxin, and T-2 triol could provide important information for evaluating animal and human exposure to residual T-2 toxin.
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Table 1 Extraction recovery rates and intraday and interday precisions of the assay for T-2 toxin, HT-2 toxin, and T-2 triol in plasma, urine, liver, kidney, and fat. Sample matrix
Analyte
Spiked concentration ng/mL (g/kg)
Extraction recovery (%)
Plasma
T-2
1 10 100 2 20 200 5 50 500
92.3 95.2 101 95.4 92.2 89.3 90.1 93.2 96.3
± ± ± ± ± ± ± ± ±
4.09 3.78 4.53 3.79 3.16 3.71 4.31 3.28 3.02
4.4 4.0 4.5 3.9 3.4 4.2 4.8 3.5 3.1
9.3 7.9 8.2 9.3 8.2 9.6 9.2 8.5 7.3
1 10 100 2 20 200 5 50 500
93.1 91.2 89.3 92.3 91.7 88.3 92.1 94.2 100
± ± ± ± ± ± ± ± ±
3.45 3.49 2.91 2.98 3.36 2.81 3.46 3.29 2.40
3.7 3.8 3.3 3.2 3.7 3.2 3.8 3.5 2.4
8.4 7.3 6.9 10.2 9.4 10.0 9.8 8.1 7.3
1 10 100 2 20 200 5 50 500
85.1 80.2 83.7 75.9 81.3 83.2 79.4 81.2 83.6
± ± ± ± ± ± ± ± ±
3.62 2.85 3.33 2.12 2.39 3.12 4.21 3.15 3.63
4.3 3.6 4.0 2.8 2.9 3.8 5.3 3.9 4.3
7.9 6.8 8.3 9.5 8.3 7.9 8.1 7.7 9.3
1 10 100 2 20 200 5 50 500
83.1 81.0 86.3 77.4 80.6 85.6 82.1 80.5 87.6
± ± ± ± ± ± ± ± ±
2.92 2.52 3.27 3.87 3.05 3.27 2.35 2.56 4.53
3.5 3.1 3.8 5.0 3.9 3.8 2.9 3.2 5.2
8.3 7.9 9.2 9.4 7.0 8.2 6.4 7.4 10.1
1 10 100 2 20 200 5 50 500
75.3 78.3 75.3 75.4 78.8 76.6 74.3 77.3 74.2
± ± ± ± ± ± ± ± ±
3.03 2.64 3.42 2.72 4.21 3.12 2.52 2.03 2.45
4.0 3.4 4.5 3.6 5.3 4.1 3.4 2.6 3.3
8.3 9.7 8.1 7.3 9.9 8.4 8.2 9.1 8.6
HT-2
T-2 triol
Urine
T-2
HT-2
T-2 triol
Liver
T-2
HT-2
T-2 triol
Kidney
T-2
HT-2
T-2 triol
Fat
T-2
HT-2
T-2 triol
3.4. Tissue distribution studies Tissue distributions of T-2 toxin, HT-2 toxin, and T-2 triol were investigated in pigs following a single i.v. dose of T-2 toxin (1 mg/kg BW). The results (Fig. 3) indicated that T-2 toxin is rapidly metabolized into more polar metabolites of HT-2 toxin and T-2 triol. They underwent a rapid and wide distribution into tissues during the examination period. This result is in accordance with the
Intra-day (RSD %)
Inter-day (RSD %)
metabolism of T-2 toxin in tissues and gastrointestinal tract of swine [11]. T-2 toxin was not detected in the liver and kidney, most probably T-2 toxin was indeed distributed into these main eliminating organs, and it was extensively metabolized or excreted. This result is consistent with the findings that T-2 toxin was not detected in the liver after arterial injection of T-2 toxin at 1.2 mg/kg BW in pigs [10] and after oral administration of 3H T-2 toxin in mice [9].
Table 2 Main toxicokinetics parameters of T-2 toxin and its metabolites (HT-2 toxin and T-2 triol) after a single i.v. administration of T-2 toxin (1 mg/kg BW) in pigs. Results are presented as means ± SD. Toxicokinetic parameters
T-2 toxin
z (1/min) t1/2z (min) AUC0–∞ (g min mL−1 ) MRT (min) Cmax (ng/mL) Vss (L/kg) CL (L/min/kg)
0.021 34.63 42.53 22.06 2736 1.18 0.025
± ± ± ± ± ± ±
0.002 2.95 4.60 1.71 236.30 0.08 0.003
HT-2 toxin
T-2 triol
0.010 ± 73.79 ± 13.13 ± 94.56 ± 208.1 ± – –
0.005 ± 159 ± 4.98 ± 211.0 ± 116.3 ± – –
0.001 5.53 2.08 5.40 25.30
0.001 13.40 0.25 19.40 14.50
Notes. AUC0–∞ : the area under the plasma concentration–time curve; t1/2z : half-life of elimination, Cmax : maximum plasma concentration; Tmax : time to maximum plasma concentration, CL: clearance; Vss : volume of distribution; MRT: mean residence time; SD: standard deviation.
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Fig. 3. Tissue distribution of T-2 toxin, HT-2 toxin, and T-2 triol after a single i.v. administration of T-2 toxin (1.0 mg/kg BW) in pigs (n = 3). Results are presented as mean values + SD.
The highest T-2 toxin concentration was detected in fat tissues (58.6 ± 8.21 g/kg), followed by the lungs (54.0 ± 4.27 g/kg) and spleen (47.8 ± 5.70 g/kg). However, Beasley et al. [10] reported that fat could not be detected in pigs, which may be attributed to factors including the age of the animals, administration dosage, and body conditions. The levels of T-2 toxin in all tissues could not be detected at 6 h after dosing. The highest HT-2 toxin concentrations were also observed in the liver (216.3 ± 16.9 g/kg), followed by the kidney (206.3 ± 20.3 g/kg) and the small intestines (140.5 ± 9.2 g/kg). HT-2 toxin was still detected at 6 h after administration. T-2 toxin, HT-2 toxin, and T-2 triol were found at low concentrations in the brain, which suggest that the toxicants did not efficiently cross the blood–brain barrier. The results of T-2 triol were similar to that of HT-2 toxin in tissues, except its concentrations were much lower than that of HT-2 toxin. In summary, T-2 toxin is metabolized into an extensive range of metabolites such as HT-2 toxin, especially in the liver, kidney, and small intestines.
Given the lower toxicity and complex elimination characteristics of the metabolites than T-2 toxin, we just obtained the elimination half-lives (t1/2z ) of T-2 toxin. The mean log-transferred concentrations (ln C) of T-2 toxin in pig tissues over time were analyzed by linear regression analysis, except for the liver and kidney. The t1/2z of T-2 toxin in tissues including fat, muscle, stomach, brain, small intestines, heart, lung and spleen were 41.24 min, 33.12 min, 25.53 min, 22.64 min, 21.20 min, 16.32 min, 15.33 min, 11.71 min, respectively. According to the results, T-2 toxin has a longer t1/2z (41.24 min) in fat tissues, indicating its selective retention. The depletion of T-2 toxin in the pig spleen was much faster than other tissues. 3.5. Excretion studies The cumulative excretion of T-2 toxin, HT-2 toxin, and T-2 triol in urine and feces after a single i.v. administration of T-2 toxin (0.5 mg/kg BW) was determined. 4 h after administration of T-2
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Fig. 4. The quality of T-2 toxin, HT-2 toxin, and T-2 triol in urine in different time after a single i.v. administration of T-2 toxin (0.5 mg/kg BW) in pigs (n = 7). Results are presented as mean values + SD.
toxin, the T-2 toxin, HT-2 toxin, and T-2 triol concentrations in urines were 30.9 ± 2.1 ng/mL, 614.4 ± 177 ng/mL, 306 ± 70 ng/mL, respectively. The excretion data of T-2 toxin, HT-2 toxin, and T2 triol in urine indicated that merely
